Abstract

We evaluated delta-9 tetrahydrocannabinol (Δ9-THC), delta-8 tetrahydrocannabinol (Δ8-THC), CP55,940 (CP55), 1-deoxy-11-hydroxy-Δ8-THC-dimethylheptyl (deoxy-HU210, a CB2-selective cannabinoid that also binds the CB1 receptor) and the endogenous cannabinoid anandamide (ANA) via i.c.v. and/or intrathecal (i.t.) routes of administration, alone and in combination with SR141716A (SR), a CB1 antagonist, using the tail-flick test. Our studies were performed in order better to characterize potential diversity in interactions of the cannabinoids with the cannabinoid (CB1) receptor. When SR was administered i.c.v. or i.p. before Δ9-THC, Δ8-THC or CP55 (i.c.v. or i.t.), SR was a potent antagonist and the blockade was complete (AD50≤ 8.1 μg/mouse i.c.v. or AD50 ≤ 1.4 mg/kg i.p.). The AD50 values (dose of antagonist that produced a 50% antagonism of agonist effects) for blockade of Δ9-THC, Δ8-THC, CP55,940 (i.c.v. or i.t.) by SR (i.c.v. or i.p.) differed significantly for only two combinations [Δ8-THC/SR, both i.c.v. and CP55 (i.t.)/SR (i.p.)]. Conversely, SR (i.t.) produced an incomplete block of the antinociceptive effects of i.t. Δ9-THC, Δ8-THC and CP55 (AD50 = 28.6, 50.2 and 20.9 μg/mouse, respectively). Blockade of the deoxy-HU210 (i.c.v.) by SR (either i.c.v. or i.p.) was incomplete and AD50 values could not be calculated. Although the maximal blockade of deoxy-HU210 (i.t.) by SR (i.t.) was only 50%, SR administered i.p. before deoxy-HU210 (i.t.) produced a potent and complete blockade (AD50 = 0.4 mg/kg). The effects of SR on ANA-induced antinociception were mixed. The maximal attenuation of the ANA (i.t.) by SR (i.t.) was 38%. SR (i.p.) blockade of ANA was complete, but the AD50 was 15.4 mg/kg, greater than 15-fold higher than that required to block Δ9-THC, Δ8-THC, CP55 or deoxy-HU210. In addition, SR (i.p. or i.t.) failed to block the hypothermic effects of ANA (i.t.), while completely reversing the hypothermic effects of Δ9-THC (i.t.). These data indicate that SR has a much greater efficacy at supraspinal than at spinal sites. Alternatively, such data suggest either a differential interaction of the cannabinoids at the CB1 receptor or the existence of subtypes of the CB1 receptor.

We have accumulated evidence indicating that cannabinoids produce antinociception by indirect interaction with kappa opioids in the spinal cord after i.t. administration (Smith et al.,1994b). The kappa antagonist nor-binaltorphimine (nor-BNI) and dynorphin antisera block Δ9-THC-induced (THC i.t.) antinociception but do not block THC-induced catalepsy, hypothermia or hypoactivity (Smith et al., 1994a; Pugh et al.,1996; Welch, 1993). In addition, the discovery of the bidirectional cross-tolerance of Δ9-THC and CP55 to kappaagonists using the tail-flick test (Smith et al., 1994a) and to dynorphin A (Welch, 1997), indicates that cannabinoids interact in a yet-to-be-determined manner with kappa opioids. The attenuation of the antinociceptive effects of THC by antisense to thekappa-1 receptor further implicates the release of endogenous kappa opioids in the mechanism of action of the cannabinoids (Pugh et al., 1995). In addition, dynorphin antibodies block cannabinoid-induced antinociception, and prevention of the metabolism of dynorphin A (1–17) to dynorphin (1–8) or to leucine enkephalin prevents the enhancement of morphine-induced antinociception by the Δ9-THC (Pugh et al., 1996).

The potent, synthetic cannabinoid CP55 was instrumental in demonstrating that cannabinoid binding sites are present in the substantia gelatinosa, an area involved with the transmission of pain signals (Herkenham et al., 1990). In addition, CP55 produces many of the behavioral and physiologic effects characteristic of THC. Despite these similarities, we have found that THC and CP55 differ in their interaction with morphine in the spinal cord (Welch and Stevens, 1992). Pretreatment of mice with CP55 (i.t.) does not enhance the antinociceptive effects of morphine (i.t.), whereas pretreatment with THC produces a 10-fold decrease in the morphine ED50. Our data indicate that THC enhances the antinociception of morphine through the release of endogenous dynorphin A (Pugh et al., 1996); CP55 appears to release dynorphin B (Pugh et al., 1997).

Given the aforementioned diversity in the antinociceptive effects of various cannabinoids, we evaluated the ability of the cannabinoid CB1 antagonist SR to attenuate the antinociceptive effects of several cannabinoids in two test systems: the tail-flick test for antinociception and rectal temperature evaluation for hypothermic effects commonly observed with cannabinoids. We evaluated the effects of SR via i.t., i.c.v. and i.p. routes of administrationvs. the cannabinoids administered either i.t. or i.c.v. The cannabinoids evaluated included Δ9-THC and Δ8-THC, which have been shown to interact with dynorphin A systems (Welch et al., 1995a; Pugh et al.,1996); CP55, which has been shown to release dynorphin B; deoxy-HU210, which has been shown to have nearly a 38-fold selectivity for the CB2 receptor (Huffman et al., 1996) and anandamide, which has been shown to fail to interact with dynorphinergic systems (Welch, 1997). Our initial goal was to determine the pA2 values for SRvs. the various cannabinoids in order to obtain some indication of potential subtypes of the CB1 receptor. However, it soon became apparent that we would not be able to perform full shifts of curves for anandamide or other cannabinoids as a consequence of only partial antagonism by SR. We have therefore presented the data as the differential AD50 values for SR vs. the various cannabinoids as an indicator of potential differences in binding of the cannabinoids at the CB1 receptor.

Materials and Methods

Animals.

Male ICR mice (Harlan Laboratories, Indianapolis, IN) with a weight range of 23 to 27 g were housed six or eight to a cage in animal care quarters maintained at 22 ± 2°C on a 12-hr light/dark cycle. Food and water were available ad libitum.

Intrathecal injections.

Intrathecal injections were performed according to the protocol of Hylden and Wilcox (1983). Unanesthetized mice were injected between the L5 and L6 areas of the spinal cord with a 30-gauge, 1/2-inch needle. Injection volumes of 5 μl were administered. Cannabinoids and SR were prepared in 100% DMSO. DMSO vehicle produced scratching behavior in mice that lasted 2 min after injection. Other vehicles have previously been tested in our laboratory. Ethanol/saline (1:10) and emulphor/ethanol/saline (1:1:18) produced significant antinociceptive effects alone in the tail-flick test and were not used as the cannabinoid vehicle when performing i.t. injections.

Intracerebroventricular injections.

Intracerebroventricular injections were performed according to the method of Pedigo et al. (1975). Mice were lightly anesthetized with ether, and an incision was made in the scalp such that the bregma was exposed. Injections were performed using a 26-gauge needle with a sleeve of PE 20 tubing to control the depth of the injection. Mice were administered an injection volume of 5 μl at a site 2 mm rostral and 2 mm caudal to the bregma at a depth of 2 mm. The cannabinoids and SR were prepared in 1:1:18 (emulphor/ethanol/saline) for i.c.v. administration. Comparison of vehicles for the cannabinoids by the i.c.v. route of administration indicated that 1:1:18 (emulphor/ethanol/saline) vehicle was devoid of antinociceptive effects (less than 10% MPE). The DMSO vehicle, which proved inactive (less than 15% MPE) upon i.t. administration, had variable effects upon i.c.v. administration (between 10% and 25% MPE) and was therefore not used for the i.c.v. route of administration.

Intraperitoneal administration of SR.

SR was dissolved in 1:1:18 (emulphor/ethanol/saline) for i.p. administration. The use of DMSO i.p. in animals leads to a long duration of abdominal irritation and abdominal scratching that interferes with the testing procedure. The 1:1:18 (emulphor/ethanol/saline) vehicle has a long history of use by many laboratories for solubilization of cannabinoids and is devoid of antinociceptive effects in our test systems.

SR time course.

A time course study of SR (i.p., i.t. and i.c.v.) block of Δ9-THC-induced (i.t. and i.c.v.) antinociception was evaluated. In all cases the peak time-point for blockade was at 1 hr. An example of one study of SR (i.p.)vs. Δ9-THC (i.t.) is shown in figure1. A similar study was performed using SRvs. anandamide, and again the peak blockade of anandamide by SR was at 1 hr. Thus the 1 hr time-point was chosen for all subsequent studies of SR in combination with the cannabinoids. Δ9-THC, Δ8-THC, deoxy-HU210 and CP55 or DMSO vehicle (i.t.) were administered 15 min before determination of the response latency of the mice in the tail-flick test. This time-point represents the peak effect of the compounds as determined in our laboratory in numerous previous studies. Anandamide in 100% DMSO was administered 3 min before testing, the time of peak antinociception (Smith et al., 1994a).

Time course of SR administered i.p. before Δ9-THC (i.t.). SR was administered i.p. at 5, 20, 40 and 60 min before THC (i.t., 100 μg/mouse). Eight mice were used per treatment group. The average %MPE was calculated for each group and compared statistically using ANOVA followed by Dunnett’s ttest. * indicates that the effect was significant at the P < .05 level.

SR blockade of cannabinoids.

The effects of SR on cannabinoid antinociception were evaluated using ED80 doses of the cannabinoids in combination with SR. Thus in all cases, the AD50 for SR blockade of cannabinoid-induced antinociception represents the dose of SR that blocks equally efficacious doses of the cannabinoids. Although the data are not shown, the SR (i.p.)-induced shifts of the dose-effect curves for Δ9-THC (i.t.), CP55 (i.t.) and Δ8-THC (i.t.) were parallel rightward shifts as evaluated by the method of Tallarida and Murray (1987). For the reference of the reader, the ED50 values CLs for Δ9-THC, CP55 and Δ8-THC (all i.t.) are 44.97 (22.96–88.09), 2.28 (0.006–8.59) and 72.07 (36.06–144), respectively. The ED50 values plus CLs for Δ9-THC, CP55 and Δ8-THC (all i.c.v.) are 16.4 (11–24.8), 2.89 (1.6–5.1) and 125.8 (65–244), respectively. ED50 values for the drugs administered i.t. do not differ significantly from the ED50 values after i.c.v. administration (Welch et al., 1995b).

Tail-flick test.

The tail-flick procedure used was that ofD’Amour and Smith (1941). Control reaction times of 2 to 4 sec and a cutoff time of 10 sec were used. Antinociception was quantified as the %MPE as developed by Harris and Pierson (1964) using the following formula:%MPE=100×[(test−control)/(10−control)]We calculated %MPE for each mouse using at least six mice per dose. By using the %MPE for each mouse, we calculated the mean effect and S.E.M. for each dose. Dose-response curves were generated using at least three doses of drug. ED50 values were determined by log-probit analysis and CLs were determined using the method ofTallarida and Murray (1987) for graded dose-response curves, omitting doses that produced 0% effect or 100% effect. The percent antagonism of an ED80 dose of drug by SR was determined using the following formula:%Antagonism=100×[average%MPE with SR pretreatment][average%MPE with vehicle pretreatment]We calculated %MPE for each mouse using at least six mice per dose. The %MPE for each mouse pretreated with SR was divided by the average %MPE for a group of six mice pretreated with vehicle. The percent antagonism for each mouse pretreated with SR was calculated. Each experiment was replicated at least twice. AD50 values were generated using at least three doses of SR by log-probit analysis, and CLs were determined with the method of Tallarida and Murray (1987)as described above, using the percent antagonism for each mouse and at least 12 mice per dose of SR. Significant differences between AD50 values were determined as a lack of overlap of the CLs for the AD50 values.

Hypothermia.

Base-line rectal temperatures were determined before drug or vehicle injection with a telethermometer (Yellow Springs Instrument Co., Yellow Springs, OH) and a thermistor probe inserted to 25 mm. Rectal temperatures were measured again after the injection. The difference between values before and after injection was calculated for each animal. Statistical analysis of all hypothermia data was performed using ANOVA with Dunnett’s t test for comparison with vehicle or Dunnett’s t test for comparisons among all groups (Dunnett, 1955).

Results

SR (i.t.) produced an incomplete block of the antinociceptive effects of i.t. Δ9-THC, Δ8-THC and CP55 (AD50 = 28.6, 50.2 and 20.9 μg/mouse, respectively) (fig.2). The AD50 values and CLs for all studies are summarized in table1. Doses of the drugs tested are listed in table 1 and represent nearly equivalent antinociceptive effects (approximate ED80 doses). Because of the partial blockade of the cannabinoids by SR (i.t.), the CLs about the AD50values are larger than for other drug administrations in which a complete block by SR was observed. Increasing the dose of SR to 100 μg/mouse failed to produce any greater blockade than that observed with 50 μg/mouse. In addition, the solubility of the drug at greater than 100 μg/mouse (20 mg/ml) was poor. SR at any dose tested failed to produce either antinociceptive or hyperalgesic effects in this test system. The maximal attenuation of the ANA ED80 (i.t.) by SR (i.t.) was 38% (fig. 2). The AD50 for SR in the presence of ANA could not be calculated, although the effects of SR led to a significant blockade of ANA.

Antagonism of the effects of cannabinoids administered i.t. by SR administered i.t. SR was administered i.t. at 1 hr before the ED80 doses of the cannabinoids (as given in table 1) or vehicle (i.t.) in mice. At 15 min later the mice were tested for antinociception using the tail-flick test, with the exception of anandamide, which was tested at 3 min after administration. Eight mice were used per treatment group. The average %MPE was calculated with the vehicle control using ANOVA followed by Dunnett’s t test. * indicates significance at the P < .05 level.

AD50 values for SR blockade of cannabinoid-induced antinociceptive effects after administration to mice

Deoxy-HU210 had not previously been tested after i.t. or i.c.v. administration. The effects of deoxy-HU210 were dose-related after both routes of administration (fig. 3) upon a peak time of testing at 15 min after administration. The ED50 values for the drug were 4.9 μg/mouse (1.5–16.4) after i.t. administration and 3.1 μg/mouse (1.6–8.2) after i.c.v. administration. The maximal attenuation of the deoxy-HU210 i.t. ED80 by SR (i.t.) was 50% attenuation (fig.4). The AD50 for SR i.t.vs. deoxy-HU210 could not be calculated, although the effects of SR led to a significant blockade of the drug.

Dose-response curves of deoxy-HU210 administered either i.t. or i.c.v. to mice. Deoxy-HU210 was administered either i.t. (■) or i.c.v. (○) to mice. At 15 min later the mice were tested for antinociception using the tail-flick test. Eight mice were used per treatment group. The average %MPE was calculated for each treatment group (± S.E.M.), and the ED50 was determined using the method of Litchfield and Wilcoxon (1949).

Antagonism of the effects of deoxy-HU210 administered (i.t. or i.c.v.) by SR (i.p., i.t. or i.c.v.). SR was administered by all routes at 1 hr before the ED80 dose of deoxy-HU210 (as given in table 1) or vehicle in mice. At 15 min later the mice were tested for antinociception using the tail-flick test. Eight mice were used per treatment group. The average %MPE was calculated for each treatment group (± S.E.M.) and compared with the vehicle control using ANOVA followed by Dunnett’s t test. * indicates significance at the P < .05 level.

However, when SR was administered i.c.v. before the cannabinoids [Δ9-THC, Δ8-THC or CP55 (i.c.v.)], SR was a potent antagonist and the blockade was complete and dose-related (fig. 5). The AD50 values for SR (i.c.v.) vs. these cannabinoids (i.c.v.) ranged from 2.4 μg/mouse to 8.1 μg/mouse (table 1). The only significant difference in AD50 was observed for SR (i.c.v.) in combination with Δ8-THC, where more than 2-fold more SR was required to block Δ8-THC. Anandamide is not active after i.c.v. administration (Smith et al., 1994a) and thus could not be tested i.c.v. in combination with SR (i.c.v.). A study of deoxy-HU210 (20 μg/mouse, i.c.v.) in combination with SR (30 μg/mouse, i.c.v.) was performed (fig. 4). The effect of SR was significant, but an incomplete block resulted; higher doses of SR failed to block deoxy-HU210 completely. Interestingly, SR at 10 and 20 μg/mouse failed to alter the antinociceptive effects of deoxy-HU210 significantly [data not shown because the effect does not differ from (i.c.v.) deoxy-HU210 alone in fig. 4]. Thus we were unable to calculate an AD50 for SR (i.c.v.) vs.deoxy-HU210 (i.c.v.).

Antagonism of the effects of cannabinoids administered i.c.v. by SR administered i.c.v. SR was administered i.c.v. at 1 hr before the ED80 doses of the cannabinoids (as given in table 1) or vehicle (i.c.v.) in mice. At 15 min later the mice were tested for antinociception using the tail-flick test. Eight mice were used per treatment group. The average %MPE was calculated for each treatment group (± S.E.M.) and compared with the vehicle control using ANOVA followed by Dunnett’s t test. * indicates significance at the P < .05 level.

The AD50 values for blockade of Δ9-THC, Δ8-THC and CP55 (i.c.v.) by SR (i.p.) also did not differ significantly (fig. 6; table 1). The AD50 values ranged from 0.47 mg/kg to 1.4 mg/kg, and the blockade by SR was complete. The blockade of deoxy-HU210 (i.c.v.) by SR (i.p.) was incomplete (48% blockade) using 50 μg/mouse SR. SR (100 μg/mouse) produced no greater effect than the 50 μg/mouse (data not shown).

Antagonism of the effects of cannabinoids administered i.c.v. by SR administered i.p. SR was administered i.p. at 1 hr before the ED80 doses of the cannabinoids (as given in table 1) or vehicle (i.c.v.) in mice. At 15 min later the mice were tested for antinociception using the tail-flick test. Eight mice were used per treatment group. The average %MPE was calculated for each treatment group (± S.E.M.) and compared with the vehicle control using ANOVA followed by Dunnett’s t test. * indicates significance at the P < .05 level.

The AD50 values for blockade of Δ9-THC, Δ8-THC and deoxy-HU210 (i.t.) by SR (i.p.) did not differ significantly from each other (figs. 4 and 7; table1) and ranged from 0.4 to 0.9 mg/kg. However, the blockade by SR (i.p.) of CP55 was significantly different in that we generated a 9-fold lower AD50. However, the AD50 for SR (i.p.) blockade of ANA (i.t.) was 15.4 mg/kg, significantly greater than 15-fold higher than that required to block Δ9-THC, Δ8-THC, CP55 and deoxy-HU210.

Antagonism of the effects of cannabinoids administered i.t. by SR administered i.p. SR was administered i.p. at 1 hr before the ED80 doses of the cannabinoids (as given in table 1) or vehicle (i.t.) in mice. At 15 min later the mice were tested for antinociception using the tail-flick test, with the exception of anandamide, which was tested at 3 min after administration. Eight mice were used per treatment group. The average %MPE was calculated for each treatment group (± S.E.M.) and compared with the vehicle control using ANOVA followed by Dunnett’st test. * indicates significance at the P < .05 level.

Antagonism of the hypothermic effects of anandamide and Δ9-THC administered i.t. by SR administered i.t. (panel A) or i.p. (panel B). SR was administered i.t. or i.p. at 1 hr before the ED80 doses of the cannabinoids (as given in table 1) or vehicle (i.t.) in mice. At 15 min later the THC-pretreated mice (filled bars) were tested for hypothermia. Mice pretreated with anandamide (white bars) were tested at 3 min after administration. Eight mice were used per treatment group. The average change in body temperature from that of naive mice was calculated for each treatment group (± S.E.M.) and compared with the vehicle control using ANOVA followed by Dunnett’s t test. * indicates significance at the P < .05 level.

Discussion

The present work is an outgrowth of our initial finding that the cannabinoids enhance the antinociceptive effects of the opioids (Welch and Stevens, 1992). The focus of this manuscript is the interaction of the cannabinoids with the CB1 receptor as quantitated by the actions of the highly CB1-selective antagonist SR (Rinaldi-Carmona et al., 1994; Felder et al., 1995; Showalter et al., 1996) to attenuate such antinociceptive effects. SR has been extensively studied in a variety of systems and appears to be selective for the CB1 receptor (Rinaldi-Carmona, 1995; Rinaldi-Carmona et al., 1996b; Showalter et al., 1996; Felder et al., 1995). Compton et al. (1996) have evaluated the effects of SR-induced blockade of a tetrad of traditional cannabinoid behaviors, in addition to the p-phenylquinone (PPQ) test for antinociception, using both i.v. and i.p. administration of SRvs. i.v. administration of Δ9-THC. They did not evaluate the effects of SR vs. any cannabinoid administered centrally. Although the time course of effects of SR (i.v.) observed by Compton et al. (1996) differs from those observed in our study (i.p., i.c.v. and i.t.), such an effect is to be expected given the differences in routes of administration of the drug. However, their AD50 for SR (i.v.) blockade of the antinociceptive effects of Δ9-THC (i.v.) [0.16 mg/kg in the tail-flick test] is within the range observed in our study for SR (i.p.) block of Δ9-THC, Δ8-THC and CP55 or deoxy-HU210 (all i.t.) (table 1). Similarly, the AD50values generated for SR (i.p.) by Compton et al. (1996)[0.38 mg/kg in the tail-flick test and 2.7 mg/kg in the PPQ test] are also in the range of those shown in our study to block Δ9-THC, Δ8-THC and CP55 (i.c.v.). These data indicate that the efficacy and potency of SR (i.p.) are similar to those of SR administered i.v. In addition, the AD50 values for SR (i.p. and i.v.) are similar to those for peripherally or centrally administered cannabinoids.

However, Compton et al. (1996) did not evaluate the block by SR of diverse cannabinoids. One major difference between the cannabinoids tested in our study was that SR (i.p.) was at least 15-fold less effective in blocking the effects of anandamide administered i.t. than in blocking the other classic cannabinoids. Because anandamide has been shown to have somewhat higher affinity for the CB1 receptor (Showalter et al., 1996) and to displace [3H]-SR binding with a Ki similar to that of Δ9-THC (Hirst et al., 1996), such a difference was unexpected and may represent some differences in the binding of anandamide to the CB1 receptor. We were not able to generate a pA2 value for SR block of anandamide, so we cannot provide evidence of a different CB1 receptor subtype for anandamide binding, although such a possibility cannot be ruled out. Judging by the lack of interaction of anandamide with the dynorphinergic system in the production of antinociception and tolerance, it appears reasonable to speculate on the existence of potential subtypes of the CB1 receptor. The CB1A receptor has been cloned (Shire et al., 1995) and characterized in cell lines (Rinaldi-Carmonaet al., 1996a), but it differs only slightly from the CB1 receptor in the events mediated by activation of the CB1Areceptor. SR has about 10-fold lower affinity at the CB1Areceptor than at the CB1 receptor. Anandamide has nearly equal affinity for both isoforms of the CB1 receptor. Given such data, the potential for other isoforms of the receptor cannot be ruled out, nor can the potential for differences in SR binding at such putative new CB1 receptor subtypes.

A similar difference between cannabinoids that we tested was observed with the CB2-selective drug deoxy-HU210 (Huffman et al.,1996). The i.p. administration of SR only partially attenuated the antinociceptive effects of deoxy-HU210 (i.c.v.). Deoxy-HU210 has high affinity at the CB1 receptor as well as at the CB2 receptor. The AD50 for SR (i.p.) vs. deoxy-HU210 (i.t.) did not differ from other cannabinoids. Thus it was surprising that the effects of the drug in combination with SR differed from other cannabinoids upon i.c.v. administration. Because at spinal sites deoxy-HU210 appears to interact with the CB1 receptor, the lack of efficacy of SR (i.c.v. or i.p.) in blocking the drug’s effects after supraspinal administration of deoxy-HU210 (i.c.v.) may simply reflect some pharmacokinetic interaction with SR. Alternatively, the data may indicate that the binding of deoxy-HU210 to the CB1 receptor supraspinally differs from that spinally or that subtypes of the CB1 receptor exist. We have no data to indicate why such a diversity in the effect of SR vs. deoxy-HU210 is observed.

Unlike work in the myenteric plexus of the guinea pig ileum, where SR was less potent in blocking contractile inhibition induced by CP55vs. Δ9-THC (Pertwee et al., 1996), we found few differences among Δ9-THC, Δ8-THC and CP55 in the AD50 values for blockade by SR via any route of administration. Our data indicated that SR (i.p.) was more potent in blocking CP55 (i.t.) and less potent in blocking Δ8-THC when both drugs were administered i.c.v. The biological relevance of such differences is not apparent, because such differences were observed as two random events and were not consistent across all the data. It is possible but unlikely that the pA2 values for such blockade differ significantly. Our data indicate that, unlike the suggestion of different cannabinoid receptors for Δ9-THC vs.CP55 in the guinea pig ileum (Pertwee et al., 1996), in our system we have only the above evidence based on the SR data to indicate differences in binding sites for Δ9-THC vs.CP55 in the spinal cord. However, we have demonstrated differential release of dynorphin A vs. dynorphin B by Δ9-THC vs. CP55, respectively (Pugh et al., 1997). It is difficult to envision such diverse dynorphin release profiles for the drugs if they exert their effects through actions at one receptor subtype. The mechanisms underlying the differential release of dynorphins by Δ9-THCvs. CP55 thus remain unknown.

SR appears to lack potency when administered at the spinal segmental level. Comparison of the AD50 values for SR (i.t.) block of cannabinoids (i.t.) with the AD50 values for SR (i.c.v.) block of cannabinoids (i.c.v.) indicates that a 6- to 9-fold increased dose of SR was required at spinal sites. In addition, the block was incomplete in all cases. Thus not only the potency, but also the efficacy, of SR is low when it is administered spinally. The AD50 for SR vs. deoxy-HU210 and for anandamide (all i.t.) could not be determined. These data indicate that the predominant effects of SR may be at supraspinal sites. Because the binding of [3H]-CP55 to presumably the CB1 receptor does not appear to differ kinetically at brain vs. spinal sites (Smith et al., 1994a; Welch et al., 1995a), and because displacement of SR binding at spinal sites has not been evaluated, there is no evidence for anandamide- or deoxy-HU210-sensitive receptor subtypes. However, this lack of evidence does not rule out such a possibility. In addition, the lack of efficacy of SR in blocking the antinociceptive effects of anandamide might be explained by SR exerting its effects predominantly on antinociception at supraspinal sites, a region where anandamide fails to alter antinociception in mice (Smith et al., 1994a; Welch et al., 1995a) and rats (Lichtman et al., 1996). Anandamide does produce a small but significant hypothermic effect when administered i.t., but not when administered in the rat brain (Lichtmanet al., 1996). Presumably, such an effect of anandamide (i.t.) would be supraspinally mediated. Δ9-THC (i.t.) produces a robust hypothermic effect when administered i.t. The hypothermic effects of Δ9-THC are blocked totally by SR; the hypothermic effects of anandamide (i.t.) are not altered by SR. Such data are indicative of differential interactions of the two cannabinoids in temperature regulation. The nature of the differential effect remains to be elucidated, but it is clearly mediated by differences in the binding to the CB1 receptor supraspinally, as evidenced by the lack of blockade of anandamide by SR. Thus anandamide appears to differ from the traditional cannabinoids in that it is not active after i.c.v. administration in several behaviors that are characteristic of cannabinoids and is either incompletely blocked or not blocked by SR in quantitation of such behaviors. Other differences between anandamide and Δ9-THC have been observed in tasks involving learning and memory (Lichtman et al., 1995), drug discrimination (Wiley et al., 1995) and modulation by agonists and antagonists of classic neurotransmitter systems (Welchet al., 1995b).

It is interesting that the cannabinoids differ in that they generally fall into two categories: those that enhance the antinociceptive effects of morphine only in the spinal cord (Δ9-THC, for example) and those that enhance the effects of morphine only in the brain (CP55, for example). We believe that our data indicate that the mechanism by which the cannabinoids produce antinociception involves dynorphin release spinally and that the “greater than additive effects” of the cannabinoids with morphine and the deltaopioid DPDPE are due to the initial release of dynorphin A peptides and the subsequent breakdown of the dynorphin A to leucine enkephalin (Pughet al., 1996). We hypothesize that the functional coupling of the mu/delta andmu/kappa receptors leads to enhanced antinociceptive effects of morphine and DPDPE by the cannabinoids. Several attempts have been made to understand how the cannabinoids produce their pharmacological effects, particularly antinociception. We envision cannabinoid-induced release of dynorphins as an indirect process due to the disinhibition of yet unknown neuronal processes. The localization of the cannabinoid receptors involved in dynorphin release are not known. We hypothesize that in the spinal cord, cannabinoids produce antinociceptive effects via the direct interaction of the cannabinoid receptor with Gi/o proteins, resulting in a decreased cAMP production (Welch et al., 1995b), as well as hyperpolarization via interaction with specific potassium channels (Deadwyler et al., 1993). Thus the cannabinoids may produce disinhibition by decreasing the release of an inhibitory neurotransmitter in dynorphinergic pathways. The net result of such an effect may be an increase in dynorphin release. The events that precede and follow the release of dynorphin remain unclear. The dynorphin is probably a modulator of other “downstream” systems (possibly substance P release or interaction with NMDA-mediated events) that culminate in antinociception upon administration of cannabinoids. What has proved intriguing is the observation that cannabinoids differ in their interactions with dynorphins (and subsequently with muand delta opioids). Δ9-THC and Δ8-THC appear to interact with the dynorphin A system (Pugh et al., 1996; Welch, 1997), whereas CP55 appears to interact with and release dynorphin B (Pugh et al., 1997), although CP55 is clearly cross-tolerant to Δ9-THC (Fanet al., 1994). Δ9-THC is not cross-tolerant to dynorphin B but is cross-tolerant to the dynorphins of the “A” type (Welch, 1997).

The most pronounced difference occurs with anandamide, which is neither blocked by the kappa antagonist nor-BNI nor cross-tolerant to any dynorphins (Smith et al., 1994a, Welch et al., 1995a, Welch, 1997), although anandamide is cross-tolerant to Δ9-THC and CP55 and displaces binding of the traditional cannabinoids (Smith et al., 1994a; Welch et al.,1995a; Devane et al., 1992). Anandamide fails to enhance the activity of any opioid and does not release dynorphin A (Welch et al., 1995a; Pugh et al., 1996; Welch, 1997). Although we have not yet evaluated deoxy-HU210 for dynorphin release, our preliminary data indicate that the drug fails to enhance the activity of mu, delta or kappa opioids (data not shown). However, its antinociceptive effect is blocked by nor-BNI. Such data appear to suggest a release of dynorphin B, rather than dynorphin A based on the work with CP55 (Pugh et al., 1997).

In summary, the CB1 antagonist SR was evaluated systematically after administration by three diverse routes in combination with centrally administered natural and synthetic cannabinoids and the endogenous cannabinoid anandamide. Our data indicate that anandamide and, to a lesser extent, deoxy-HU210 appear to differ from other cannabinoids tested either in that the blockade by SR was partial or in that SR was significantly less potent in such blockade. SR failed to block the hypothermic effects induced by anandamide, while attenuating those of Δ9-THC. The potency of SR in blocking Δ9-THC did not differ from its potency in blocking CP55, although the drugs exhibit pronounced diversity in the interaction with dynorphinergic systems. Such data suggest either a differential interaction of anandamide vs. the classic cannabinoids at the CB1 receptor or the existence of subtypes of the CB1 receptor.

Acknowledgments

The authors wish to thank Dr. Billy R. Martin, Department of Pharmacology and Toxicology, Medical College of Virginia, for his assistance and collaboration on this project.